Toxins produced by Corynebacterium diphtheriae (diphtheria toxin) and Pseudomonas aeruginosa (exotoxin A) both result in host cell death by the inhibition of protein synthesis. Both of these toxins ADP ribosylate a unique diphthamide residue, produced by the post- translational modification of a specific histidine in the eukaryotic translation Elongation Factor 2 (eEF2). Recent structural analysis of eEF2 from the yeast Saccharomyces cerevisiae, which is also modified by these toxins, demonstrates the diphthamide residue lies on the tip of domain IV of the protein. This tip is proposed by cryo-electron microscopic studies to be positioned near the mRNA in the ribosome. eEF2 mediates the translocation step of protein synthesis, where the newly formed peptidyl-tRNA is moved from the A- to the P-site of the ribosome and the mRNA is moved by three bases. As such, ADP-ribosylation of this site likely affects this key function of the protein. Surprisingly, even though eEF2 has been known to be the sole target for these toxins since the 1970s, the mechanism of inhibition remains unknown. Developing countries still experience outbreaks of Diphtheria, concerns regarding re-immunization and continued protection against C. diphtheriae are emerging, and P. aeruginosa infections are observed in immunocompromised patients. Understanding the mechanism of toxicity, and potentially utilizing this information to develop drugs to prevent the effects of ADP-ribosylation of eEF2, has important biomedical implications. Our recent structural, genetic and biochemical studies have led to hypotheses on the role of domain IV and this modification in inhibiting translation. We have demonstrated dominant resistance to diphtheria toxin in the presence of ADP-ribosylated eEF2, which has led to the development of a novel system to explore the mechanism of inhibition of eEF2 by these toxins in vivo. We will test the hypothesis that ADP-ribosylated eEF2 compromises translocation function at the ribosome, and perhaps also affects fidelity. We will utilize eEF2 mutants in domain IV, near the site of ADP-ribosylation, and ADP-ribosylated eEF2 to test the consequences of toxin activity in vivo and in vitro. We will utilize the advantages of the yeast genetic system to perform focused genetic screens coupled with biochemical analyses to determine the ribosome components that are required for the inhibition of translation by ADP-ribosylated eEF2 in vivo. With yeast, novel integrated genetic, biochemical and molecular biological approaches to the study of the mechanism of toxicity and resistance to diphtheria and related toxins may provide the basis for the design of approaches to counteract the effects of these toxins in vivo. PUBLIC HEALTH RELEVANCE: Corynebacterium diphtheriae and Pseudomonas aeruginosa each produce toxins, diphtheria toxin (DT) and exotoxin A (ETA) respectively, whose activity results in host cell death. The only cellular target of these toxins is the essential eukaryotic translation Elongation Factor 2 (eEF2). In the developing world Diphtheria remains a significant health concern, even with vaccination campaigns. In a recent report from Thailand, diphtheria cases were reported throughout the 1990s, consistent with the findings that 25% of 20-39 year olds and 14% of 10-19 year olds lacked immunity to diphtheria. In Russia and neighboring country outbreaks in the 1990s, including more than 115,000 cases and 3,000 deaths from 1990 to 1997 in Russia, were mostly among adults. The resurgence of Diphtheria indicates a greater understanding of its mechanism of toxicity and prevention of the effects of infection on unimmunized individuals remains important. Infections with P. aeruginosa are a particular concern for a subset of patients such as those with cancer, cystic fibrosis or reduced immune function, in particular as multidrug-resistant strains have emerged. Surprisingly, the mechanism by which ADP ribosylation of a unique diphthamide residue on eEF2 inhibits translation and thus results in cell death remains unknown. We propose to utilize a unique genetic system in the yeast Saccharomyces cerevisiae to understanding the mechanism by which the toxic effects of these microbial toxins occurs, and in the long term to develop strategies to reduce the morbidity and mortality associated with the activity of these toxins in the cell.